醛固酮对介导大鼠肝星状细胞收缩的非钙离子依赖信号通路的影响

目的 探讨醛固酮(Aldo)对介导大鼠肝星状细胞(HSC)收缩的非Ca2+依赖性信号传导通路的影响.方法 对HSC-T6细胞给予Aldo 10 μmol/L处理,用聚硅酮膜法检测HSC-T6细胞的收缩性;在激光共聚焦显微镜下动态观察HSC-T6细胞胞内游离钙离子浓度的变化.RT-PCR检测Aldo受体阻断剂安体舒通、蛋白激酶C特异性抑制剂Stauro、Rho激酶特异性抑制剂Y27632、肌球蛋白轻链激酶特异性抑制剂ML-7对Rho-Rock通路中Rock2、RhoAGTP、RhoGEFmRNA表达水平的影响.对各组间数据行单因素方差分析,两两比较采用LSD法.结果 Aldo可诱导HSC-T6细胞收缩;Aldo对HSC-T6细胞胞内游离钙离子浓度变化无明显影响.Aldo可诱导Rock2、RhoAGTP、RhoGEF mRNA的表达增强(0.770±0.049、0.960±0.096、0.180±0.006,P值均<0.01),而其阻断剂安体舒通可明显抑制这三种元件mRNA的表达(0.440±0.166、0.370±0.180、0.050±0.001,P值均<0.01).Aldo+Y27632组上述三种元件mRNA的表达较Aldo组减弱.Aldo+ML-7+Stauro组三种元件的mRNA表达水平(0.940±0.066、1.330±0.192、0.160±0.007)较对照组(0.140±0.023、0.540±0.111、0.110±0.012)增强(P<0.05),Aldo+Y27632+ML-7+Stauro组RhoGEF(0.290±0.004,P<0.01)的表达较ML-7+Stauro两者联合抑制组(0.160±0.007)增强.结论 Aldo可诱导HSC的收缩,这与Rho激酶介导的非Ca2+依赖性信号通路相关.

Abstract：

Objective To investigate the mechanisms of Aldosterone stimulating hepatic stellate cells(HSCs) contraction via Ca2+-independent pathways. Methods HSC-T6 cell line was pre-disposed with Aldo 10 μmol/L. The cell contraction was detected by silicone-rubber-membrane cultivation directly. The concentration variation of intracellular free calcium in rat HSC was observed by laser confocal microscopy.Besides, HSC-T6 cell line was under pre-disposal treatment with the blocking agents of Aldo receptor -antisterone, protein kinase C (PKC) special blocking agent-Stauro, Rho kinase blocking agent-Y27632 and MLCK special blocking agent-ML-7 respectively prior to stimulation with aldosterone. RT-PCR was used to detect the expression of Rock2, RhoAGTP and RhoGEF in Ca2+- independent pathways mediated by Rho-kinase. Results Aldo could induce HSCs contraction. The concentration of intracellular free calcium in rat HSCs had no change after pre-disposal treatment with Aldo. The mRNA expressions of Rock2, RhoAGTP and RhoGEF increased significantly after treatment with Aldo (0.770 ± 0.049, 0.960 ± 0.096, 0.180 ±0.006, P < 0.01).When inhibited with antisterone, the mRNA expressions of the three elements were (0.440 ± 0.166, 0.370 ± 0.180 and 0.050 ± 0.001, P < 0.01), lower than that of Aldo group, but higher in ML-7+Stauro + Aldo groups (0.940 ± 0.066, 1.330 ±0.192 and 0.160 ± 0.007, P < 0.05) as compared to the control group (0.140 ± 0.023,0.540 ± 0.111 and 0.110 ± 0.012). In the Y27632 + ML-7 + Stauro+Aldo group, the mRNA expression of RhoGEF (0.290 ± 0.004, P < 0.01)was higher than that of the ML-7 + Stauro + Aldo group (0.160 ± 0.007). Conclusion Aldo could induce HSCs contraction via Ca2+-independent pathways and Rho-Rock pathway involved in the process.

Aldosterone stimulates hepatic stellate cells contraction via Ca2+-independent pathways

Objective To investigate the mechanisms of Aldosterone stimulating hepatic stellate cells(HSCs) contraction via Ca2+-independent pathways. Methods HSC-T6 cell line was pre-disposed with Aldo 10 μmol/L. The cell contraction was detected by silicone-rubber-membrane cultivation directly. The concentration variation of intracellular free calcium in rat HSC was observed by laser confocal microscopy.Besides, HSC-T6 cell line was under pre-disposal treatment with the blocking agents of Aldo receptor -antisterone, protein kinase C (PKC) special blocking agent-Stauro, Rho kinase blocking agent-Y27632 and MLCK special blocking agent-ML-7 respectively prior to stimulation with aldosterone. RT-PCR was used to detect the expression of Rock2, RhoAGTP and RhoGEF in Ca2+- independent pathways mediated by Rho-kinase. Results Aldo could induce HSCs contraction. The concentration of intracellular free calcium in rat HSCs had no change after pre-disposal treatment with Aldo. The mRNA expressions of Rock2, RhoAGTP and RhoGEF increased significantly after treatment with Aldo (0.770 ± 0.049, 0.960 ± 0.096, 0.180 ±0.006, P < 0.01).When inhibited with antisterone, the mRNA expressions of the three elements were (0.440 ± 0.166, 0.370 ± 0.180 and 0.050 ± 0.001, P < 0.01), lower than that of Aldo group, but higher in ML-7+Stauro + Aldo groups (0.940 ± 0.066, 1.330 ±0.192 and 0.160 ± 0.007, P < 0.05) as compared to the control group (0.140 ± 0.023,0.540 ± 0.111 and 0.110 ± 0.012). In the Y27632 + ML-7 + Stauro+Aldo group, the mRNA expression of RhoGEF (0.290 ± 0.004, P < 0.01)was higher than that of the ML-7 + Stauro + Aldo group (0.160 ± 0.007). Conclusion Aldo could induce HSCs contraction via Ca2+-independent pathways and Rho-Rock pathway involved in the process.